Infection due to Penicillium marneffei (renamed to Talaromyces marneffei in 2011) has emerged as an important public health problem over the past two decades due to the arrival of the HIV epidemic in Asia. Since 2004, P. marneffei has become the second most common pathogen isolated from routine blood culture, after Cryptococcus neoformans, at the Hospital for Tropical Diseases in Ho Chi Minh City, the largest referral centre for HIV care in southern Vietnam. The clinical epidemiology of P. marneffei infection has not been studied in Vietnam. The fundamental epidemiological questions regarding the pathogen reservoirs and risks of acquisition remain poorly understood. The diagnosis relies on isolation of the pathogen from clinical specimens and can take up to 14 days to identify, resulting in delayed initiation of therapy which is associated with worse treatment outcomes. This thesis aims to increase knowledge and understanding of the clinical and environmental epidemiology of P. marneffei infection and to improve the speed and accuracy of diagnosis of P. marneffei infection. The Précis provides a brief background and rationale for the thesis. Chapter 1 is an introductory chapter and provides an overview of the epidemiology, ecology, mycology, pathology, immunology, clinical features, diagnosis, and treatment of P. marneffei infection. Chapter 2 summarizes the incidence and features of P. marneffei admissions at the Hospital for Tropical Diseases in Ho Chi Minh City over a 13 year period. During this period, 795 patients with P. marneffei infection were identified and hospital charts were obtainable for 513 (65%) patients. The data showed clear seasonality with an increase in incidence of approximately 30% during the rainy season compared to the dry season. The clinical and microbiological features and treatment outcomes of the patients were characterised. Poor outcome, defined as death or worsening disease at hospital discharge, occurred in 28% of patients. History of injection drug use, shorter duration of illness, absence of fever or skin lesions, higher respiratory rates, and lower platelet counts independently predicted poor outcome. Chapter 3 describes an analysis of meteorological factors that determine penicilliosis incidence in Ho Chi Minh City. Humidity, rather than precipitation, was the most important factor that governs the seasonality of penicilliosis. Higher humidity was associated with increased odds of penicilliosis versus cryptococcosis admissions. The infection incubation period was estimated to be between one and three weeks. Chapter 4 describes an analysis of exposure and behavioural risk factors for penicilliosis based on a matched case control study of 205 culture-confirmed HIV-infected penicilliosis cases and 405 HIV-infected controls recruited from two major HIV referral centres in Hanoi and Ho Chi Minh City. Penicilliosis was independently associated with proximity or exposure to tropical plants and exposure to farmed animals. The geographical analysis showed that patients living in or traveling to the highland regions were at increased risk for penicilliosis in southern Vietnam. Chapter 5 describes the development of a Taqman real-time PCR assay based on a novel Mp1 gene target unique to P. marneffei for rapid detection of P. marneffei infection in patient plasma. The assay was tested in 70 plasma samples from HIV-infected patients (50 with culture-confirmed penicilliosis, 20 with other opportunistic infections) and showed a clinical specificity of 100% (20/20) and sensitivity of 70.4% (19/27) and 52.2% (12/23) prior to and within 24-48 hours of antifungal therapy administration, respectively. Chapter 6 is an overview discussion interpreting the implications of the major findings and the future direction of P. marneffei research. The work of this thesis increases knowledge of the clinical epidemiology of P. marneffei infection in Vietnam, providing essential data for the design of prospective studies to improve the diagnosis and treatment of P. marneffei infection in Asia. The data suggest that multiple environmental factors including humidity, tropical plants, farmed animals, and highland location, are important drivers of P. marneffei infection in southern Vietnam. The real-time PCR assay showed potential as a rapid ‘rule-in' test for P. marneffei in this pilot study and should be prospectively evaluated in a large cohort to determine if it can improve diagnostic speed and crucially, impact patient outcomes. Prevention, diagnosis and elimination all require further research to reduce the high mortality following clinical disease caused by P. marneffei in Asia.

Thesis (PhD)--Stellenbosch University, 2012. / Please refer to full text for abstract.

Made available in DSpace on 2014-06-12T18:07:11Z (GMT). No. of bitstreams: 2 arquivo7922_1.pdf: 2006759 bytes, checksum: e658b40f8129b04ae2bab24568ef6746 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / As blendas poliméricas compostas por polímeros sintéticos e amido vêm ganhando importância na área de materiais por apresentarem boas propriedades e maior biodegradabilidade quando descartadas no meio ambiente. O presente trabalho teve por objetivo avaliar a capacidade biodegradadora de uma cultura mista dos fungos Talaromyces wortmannii e Phanerochaete chrysosporium numa blenda biodegradável de polietileno de baixa densidade (PEBD)/Amido, com aplicação na indústria de embalagens. A princípio foi feita uma seleção do tipo de amido pela avaliação do crescimento dos microrganismos. Em seguida, a blenda foi obtida pela mistura de PEBD/Amido 80/20% m/m num reômetro HAAKE a temperatura de 140oC por 10 min. O material foi exposto à cultura mista por um período de 165 dias. Os níveis de biodegradação dos filmes foram caracterizados antes e após a inoculação dos fungos através de análises de variação de massa, calorimetria diferencial exploratória-DSC, microscopia eletrônica de varredura-SEM e determinação da resistência à tração na ruptura e alongamento. Ainda foi realizado um monitoramento da produção de CO2 no processo de biodegradação por 90 dias, e um estudo cinético do crescimento dos fungos em meio Sabouraud modificado com amido. Os resultados mostraram que o amido do tipo anfótero (Foxhead®5901) apresentou um bom desempenho como componente para esse tipo de blenda. O consumo crescente e gradativo da blenda como fonte de carbono alternativa ficou evidente no acompanhamento da produção de CO2. Houve um acréscimo na massa dos filmes após exposição aos fungos pela sua impregnação no material. As análises de DSC mostraram que a processabilidade do PEBD não foi alterada com a adição de amido. O material apresentou uma significativa perda em propriedades mecânicas após contato com os fungos que, segundo evidenciado na SEM, consumiram o amido presente na matriz polimérica, gerando vazios e permitindo uma reorganização de sua estrutura. O fungo Phanerochaete chrysosporium apresentou como parâmetros cinéticos μmáx (h-1) = 0,10 e KS (g/L) = 8,50, enquanto que o fungo Talaromyces wortmannii μmáx (h-1) = 0,04 e KS (g/L) = 0,18, mostrando o quanto pode variar o comportamento de duas espécies de fungos distintas crescendo sobre as mesmas condições

No description available.

No description available.

Os fungos de decomposição parda são eficientes na degradação dos polissacarídeos da madeira, pois, além das enzimas hidrolíticas, podem gerar, via reação de Fenton, radicais hidroxila que são responsáveis pela rápida despolimerização dos polissacarídeos da parede celular sem a remoção prévia da lignina. O presente trabalho analisou se enzimas hidrolíticas do fungo de decomposição parda Gloeophyllum trabeum e as endoglucanases comerciais do ascomiceto Talaromyces emersonii apresentam efeito sinérgico com celulases comerciais durante a hidrólise enzimática de substratos com teor elevado de lignina. Os substratos em questão corresponderam a bagaço de cana pré-tratado por um processo quimiomecânico que emprega sulfito alcalino. Dois níveis de tratamento foram obtidos a partir de cargas diferenciadas de sulfito de alcalino. A carga mais elevada foi de 10 g de Na2SO3 e 5 g de NaOH para cada 100g de bagaço e gerou um substrato de baixa recalcitrância. O emprego de uma carga de sulfito alcalino diminuída à metade da carga anterior gerou um substrato de elevada recalcitrância. Para viabilizar a execução dos experimentos utilizando pequenas quantidades de enzima, foi desenvolvido um método de hidrólise em pequena escala empregando bagaço de cana pré-tratado com sulfito alcalino como substrato. O método em questão empregou 20 mg de substrato e 1 mL de volume final de reação gerando dados com boa reprodutibilidade e níveis de conversão de celulose e xilana similares aos observados em ensaios tradicionais que empregam 1 g de substrato e 50 mL de meio reacional. O fungo G. trabeum foi cultivado com 7 diferentes fontes de carbono, sendo detectada a atividade de endoglucanase em todos os meios e alcançando níveis mais elevados nos cultivos com carboximetilcelulose, que foi, portanto, utilizado para produção das enzimas de G. trabeum. Um extrato de cultivo de G. trabeum foi parcialmente purificado, gerando um extrato com 2,16 UI de endoglucanases/mL e um teor de proteínas de 1,34 mg/mL. O uso das enzimas de G. trabeum e de T. emersonii em misturas com a Celluclast foi baseado nas cargas de endoglucanases totais, visto que a atividade em papel de filtro não foi detectável nos dois complexos enzimáticos. Para as reações de hidrólise do substrato que apresentava baixa recalcitrância, as conversões máximas de celulose e xilana foram da ordem de 80% após 72h de reação com cargas de endoglucanases de Celluclast de 120 UI/g de substrato. A mistura de enzimas que empregaram metade desta carga de endoglucanase advinda de Celluclast e outra metade de extratos de G. trabeum proporcionaram eficiências de hidrólise similares às obtidas com a mesma carga advinda somente de Celluclast. No caso da suplementação das misturas com endoglucanases de T. emersonii, as eficiências de hidrólise foram menores do que aquelas obtidas somente com Celluclast. As reações de hidrólise do substrato de elevada recalcitrância proporcionaram conversões de celulose e xilana máximas da ordem de 50% quando se empregou Celluclast como fonte de enzimas. A suplementação de parte das endoglucanases desta preparação por endoglucanases provenientes do extrato de G. trabeum ou de T. emersonii não favoreceram a hidrólise dos polissacarídeos presentes no substrato. / Brown-rot fungi can degrade wood polysaccharides in an efficient manner because they produce hydrolytic enzymes and also hydroxyl radicals via Fenton reaction. These systems are responsible for a rapid polysaccharide depolymerization without previous lignin removal from the wood cell walls. The present work evaluated the hydrolytic enzymes produced by the brown-rot fungus Gloeophyllum trabeum and by the ascomycete Talaromyces emersonii in mixtures with commercial cellulases to hydrolyze lignin-rich substrates. The evaluated substrates were sugar cane bagasse pretreated in a chemithermomechanical process that used alkaline sulfite in the reaction media. Two levels of pretreatment were employed by using varied loads of chemicals during the cooking step. A low recalcitrance material was prepared by pretreating the sugar cane bagasse with 10 g of Na2SO3 and 5 g of NaOH per 100g of sugar cane bagasse. Using the half of this chemical loading resulted in a second substrate that was more recalcitrant. A small scale enzymatic hydrolysis procedure was set to permit the use of low enzymes dosage in the polysaccharide hydrolyses studies. This procedure used only 20 mg of lignocellulosic substrate in 1 mL of final reaction volume. Data obtained for cellulose and xylan conversion presented good reproducibility and average conversion values very similar to that obtained in traditional experiments that use 1 g of substrate in 50 mL of reaction medium. G. trabeum was cultured in 7 different carbon sources and endoglucanase activities were detected in all cases. The highest endoglucanases levels were obtained in cultures with carboxymethylcellulose as carbon source. One culture extract from this fungus was partially purified yielding a purified extract with 2.16 IU of endoglucanases/mL and a protein content of 1.34 mg/mL. The use of the enzymes from G. trabeum and T. emersonii extracts in mixtures with commercial Celluclast was based on the total loads of endoglucanases, since filter paper activities were not detectable in the extracts obtained from the studied fungi. Cellulose and xylan from the low recalcitrance substrate were converted at yields of 80% after 72 h of hydrolysis by Celluclast loaded at 120 UI of endoglucanases/g de substrate. Using enzyme mixtures with the same endoglucanase dosage but divided in 50% from Celluclast and 50% from G. trabeum extract yielded almost the same hydrolysis efficiency. In the case of the enzyme mixtures in which the extract from T. emersonii was used instead the extract from G. trabeum the hydrolysis efficiency was lower than that obtained by the treatment with Celluclast only. Cellulose and xylan from the low recalcitrance substrate were converted at yields of 50% after 72 h of hydrolysis by Celluclast. The replacement of this endoglucanase load with endoglucanases from G. trabeum or T. emersonii extracts resulted in lower hydrolysis efficiency.

La grande majorité des colorants alimentaires naturels, utilisés dans la formulation des aliments et des boissons, proviennent des pigments extraits de matières premières végétales. Plusieurs couleurs dérivées de plantes peuvent entraîner des problèmes de formulation. Des facteurs, comme par exemple, la région, le climat, l'environnement, la variété cultivée, ont un effet de nuances de couleurs, de résistance et surtout de stabilité dans le produit final. Par ailleurs, les champignons filamenteux du genre Monascus, Penicillium et Talaromyces sont connus comme d'excellents producteurs de pigments rouges. Ces pigments intéressent de ce fait les industries car ils sont stables, non-toxiques et peuvent être utilisés comme colorants alimentaires.La recherche présentée dans le cadre de cette thèse de doctorat concerne la description des propriétés du pigment rouge que produit la souche de Talaromyces albobiverticillius isolée du milieu marin tropical autour de l'île de La Réunion. Les plans d’expérience (DOE) et la méthodologie des surfaces de réponses (RSM) ont été utilisés pour optimiser les conditions de culture et la formulation du milieu de fermentation, dans le but d'accroître les teneurs en polykétides colorés. Douze structures différentes ont été identifiées dans des extraits intracellulaires et extracellulaires des cultures fongiques, à l'aide de séparations et d'analyses spectroscopiques (HPLC-PDA-ESI/MS et RMN). Les pigments N-thréonine-monascorubramine, N-glutaryl-rubropunctamine et PP-O figurent ainsi parmi les 12 composants.Avec la demande croissante de composés colorés naturels dans le secteur industriel, les champignons isolés du milieu marin semblent présenter de nombreux intérêts. Des essais ont ainsi été menés afin d'étudier 1) l'amélioration des conditions de fermentation en fioles agitées ou en fermenteur de 2 litres; 2) les effets de la teneur en sel marin sur la synthèse des pigments; 3) des méthodes d'extraction respectueuses de l'environnement. Globalement, ces résultats font ressortir le grand potentiel des champignons marins produisant ce colorant rouge et la possibilité d'obtenir les colorants alimentaires adaptés. / It is well known that the vast majority of food colorants used in food and beverage applications comes from the pigments synthesized by plant materials. Besides, stability of many plant-derived colors can create formulation problems. Factors such as the region, the climate, the environment, the cultivar all impact colors shade, strength and overall stability in the final product. As an alternate, fungi of the genus Monascus, Penicillium and Talaromyces are known as excellent producers of red pigments. These red pigments are of industrial interest as they are stable and non-toxic and can be used as food colorants.This present research deals with the selection of high throughput red pigment producing Talaromyces albobiverticillius as a source of polyketide based natural food colorants. Design of Experiments (DoE) and Response Surface Methodology (RSM) have been used to optimize culture conditions and media formulation of fermentation process. Using Box Behnken Design (BBD), the influence of different physical factors on pigment and biomass production was studied using potato dextrose broth as culture media. The best optimal conditions were found to be with initial pH of 6.4, temperature of 24 °C, agitation speed of 164 rpm and fermentation time of 149 h gave 47.93 ± 0.58 mg /L of orange pigment, 196.28 ± 0.76 mg / L of red pigment and 12.58 ± 0.41 g /L of dry biomass. With the application of Plackett- Burman Design (PBD), 16 different media formulations were optimized using various carbon and nitrogen sources. When Sucrose and Yeast extract was used as a basal medium at 24° C, high pigment yield was observed: 695.93 ± 0.29 mg /L of orange pigment, 738.28 ± 0.51 mg / L of red pigment and 6.80 ± 0.37 g /L of dry biomass.Twelve different compounds were detected from the HPLC-PDA-ESI/MS analysis of intracellular and extracellular pigmented extracts. In particular, N-threonine-monascorubramine, N-glutaryl-rubropunctamine and PP-O were tentatively identified among these twelve compounds; further, this work reports for the first time on the PDA, MS and NMR characterization of the here named as N-GABA-monascorubramine derivative (6-[(Z)-2-Carboxyvinyl]-N-GABA-monascorubramine) pigment bearing a cis configuration at the C10-C11 double bond, in Talaromyces albobiverticillius 30548. Attempts were made to study the effects of sea salts on pigment synthesis; sustainable green extraction methods for pigments; upscaling of fermentation from shake flasks to laboratory fermenter. All these experiments with their results were discussed briefly as individual chapters. Overall, these findings bring out the potential of marine-derived red pigment producing fungi and its possibility of obtaining tailor made food colorants.

La maladie de la tache noire affecte les fruits d’ananas mature, les rendant impropre à la consommation. Actuellement, aucune méthode de contrôle n'est disponible pour cette maladie. Une meilleure connaissance du pathosystème est nécessaire pour trouver des moyens de lutte efficaces.Des entretiens et échantillonnages menés auprès de producteurs durant l’hiver austral révèlent une prévalence de la maladie de 74%. Les champignons pathogènes appartiennent à plusieurs espèces : Fusarium ananatum (72% des isolats), Talaromyces stollii (21%), F. oxysporum (6%) et F. proliferatum (1%). Leur potentiel toxinogène a été déterminer, Les champignons du genre Fusarium ont produit des mycotoxines identifiées comme les fumonisines FB1, FB2 et la beauvericine. Sur un milieu de culture ananas, une concentration en beauvericine de 34959 µg kg-1 a été mesurée pour l’espèce F. proliferatum.Une méthode d’inoculation de Fusarium ananatum directement dans le parenchyme a permis de décrire la réponse du biochimique du fruit. La voie des phénylpropanoïdes est sollicitée, particulièrement avec l’élicitation du caffeoylisocitrate et du coumaroylisocitrate dans la zone infectée. Une comparaison des profils métaboliques montre que la réponse du fruit à une inoculation est plus importante chez le cultivar résistant ‘MD-2’ que chez le cultivar sensible ‘Queen’. La majorité des métabolites élicités par l’attaque sont déjà présents dans les fruits sains mature de la variété résistante. Le potentiel antifongique des composés phénoliques à était évalué. Les acides coumarique, caféoylquinique et férulique inhibent la croissance du mycélium à des concentrations similaires à celle trouvées dans les fruits infectés.Une approche par imagerie a permis de décrire l’anatomie des fruits des deux cultivars et notamment la fusion imparfaite des sépales et bractée chez ‘Queen’. Les nectaires et les parois carpellaires jouent un rôle clef dans le processus d'infection et de colonisation de Fusarium ananatum. / Fruiltet core rot (FCR) disease affects the fruits of mature pineapples. Current disease controls are not available. A deeper knowledge of the pathosystem is needed to find an effective means of control FCR.A diagnostic survey conducted with producers during the southern winter revealed a prevalence of the disease of 74%. Pathogenic fungi belong to several species: Fusarium ananatum (72% isolates), Talaromyces stollii (21%), F. oxysporum (6%) and F. proliferatum (1%). Their toxinogenic potential was determined. Fusarium fungi produced mycotoxins identified as fumonisins FB1, FB2 and beauvericin. On a pineapple culture medium, a concentration of beauvericine of 34959 μg kg-1 was measured for the species F. proliferatum.A method of inoculating Fusarium ananatum directly into the parenchyma allowed to describe the biochemical response of the fruit. The phenylpropanoids pathway is involved, particularly with the elicitation of caffeoylisocitrate and coumaroylisocitrate in the infected zone. A comparison of the metabolic profiles shows that the response to inoculation of resistant cultivar 'MD-2' is higher than in the sensitive cultivar 'Queen'. Most of the metabolites elicited by the attack are already present in healthy mature fruits of the resistant variety. The antifungal potential of the phenolic compounds was evaluated. Coumaric, caffeoylquinic and ferulic acids inhibit mycelial growth at concentrations similar to those found in infected fruits.An imaging approach allowed to describe the anatomy of the fruits of the two cultivars, to identify the key roles played by nectaries and carpel margins in the infection and colonization process of Fusarium ananatum.

Os fungos de decomposição parda são eficientes na degradação dos polissacarídeos da madeira, pois, além das enzimas hidrolíticas, podem gerar, via reação de Fenton, radicais hidroxila que são responsáveis pela rápida despolimerização dos polissacarídeos da parede celular sem a remoção prévia da lignina. O presente trabalho analisou se enzimas hidrolíticas do fungo de decomposição parda Gloeophyllum trabeum e as endoglucanases comerciais do ascomiceto Talaromyces emersonii apresentam efeito sinérgico com celulases comerciais durante a hidrólise enzimática de substratos com teor elevado de lignina. Os substratos em questão corresponderam a bagaço de cana pré-tratado por um processo quimiomecânico que emprega sulfito alcalino. Dois níveis de tratamento foram obtidos a partir de cargas diferenciadas de sulfito de alcalino. A carga mais elevada foi de 10 g de Na2SO3 e 5 g de NaOH para cada 100g de bagaço e gerou um substrato de baixa recalcitrância. O emprego de uma carga de sulfito alcalino diminuída à metade da carga anterior gerou um substrato de elevada recalcitrância. Para viabilizar a execução dos experimentos utilizando pequenas quantidades de enzima, foi desenvolvido um método de hidrólise em pequena escala empregando bagaço de cana pré-tratado com sulfito alcalino como substrato. O método em questão empregou 20 mg de substrato e 1 mL de volume final de reação gerando dados com boa reprodutibilidade e níveis de conversão de celulose e xilana similares aos observados em ensaios tradicionais que empregam 1 g de substrato e 50 mL de meio reacional. O fungo G. trabeum foi cultivado com 7 diferentes fontes de carbono, sendo detectada a atividade de endoglucanase em todos os meios e alcançando níveis mais elevados nos cultivos com carboximetilcelulose, que foi, portanto, utilizado para produção das enzimas de G. trabeum. Um extrato de cultivo de G. trabeum foi parcialmente purificado, gerando um extrato com 2,16 UI de endoglucanases/mL e um teor de proteínas de 1,34 mg/mL. O uso das enzimas de G. trabeum e de T. emersonii em misturas com a Celluclast foi baseado nas cargas de endoglucanases totais, visto que a atividade em papel de filtro não foi detectável nos dois complexos enzimáticos. Para as reações de hidrólise do substrato que apresentava baixa recalcitrância, as conversões máximas de celulose e xilana foram da ordem de 80% após 72h de reação com cargas de endoglucanases de Celluclast de 120 UI/g de substrato. A mistura de enzimas que empregaram metade desta carga de endoglucanase advinda de Celluclast e outra metade de extratos de G. trabeum proporcionaram eficiências de hidrólise similares às obtidas com a mesma carga advinda somente de Celluclast. No caso da suplementação das misturas com endoglucanases de T. emersonii, as eficiências de hidrólise foram menores do que aquelas obtidas somente com Celluclast. As reações de hidrólise do substrato de elevada recalcitrância proporcionaram conversões de celulose e xilana máximas da ordem de 50% quando se empregou Celluclast como fonte de enzimas. A suplementação de parte das endoglucanases desta preparação por endoglucanases provenientes do extrato de G. trabeum ou de T. emersonii não favoreceram a hidrólise dos polissacarídeos presentes no substrato. / Brown-rot fungi can degrade wood polysaccharides in an efficient manner because they produce hydrolytic enzymes and also hydroxyl radicals via Fenton reaction. These systems are responsible for a rapid polysaccharide depolymerization without previous lignin removal from the wood cell walls. The present work evaluated the hydrolytic enzymes produced by the brown-rot fungus Gloeophyllum trabeum and by the ascomycete Talaromyces emersonii in mixtures with commercial cellulases to hydrolyze lignin-rich substrates. The evaluated substrates were sugar cane bagasse pretreated in a chemithermomechanical process that used alkaline sulfite in the reaction media. Two levels of pretreatment were employed by using varied loads of chemicals during the cooking step. A low recalcitrance material was prepared by pretreating the sugar cane bagasse with 10 g of Na2SO3 and 5 g of NaOH per 100g of sugar cane bagasse. Using the half of this chemical loading resulted in a second substrate that was more recalcitrant. A small scale enzymatic hydrolysis procedure was set to permit the use of low enzymes dosage in the polysaccharide hydrolyses studies. This procedure used only 20 mg of lignocellulosic substrate in 1 mL of final reaction volume. Data obtained for cellulose and xylan conversion presented good reproducibility and average conversion values very similar to that obtained in traditional experiments that use 1 g of substrate in 50 mL of reaction medium. G. trabeum was cultured in 7 different carbon sources and endoglucanase activities were detected in all cases. The highest endoglucanases levels were obtained in cultures with carboxymethylcellulose as carbon source. One culture extract from this fungus was partially purified yielding a purified extract with 2.16 IU of endoglucanases/mL and a protein content of 1.34 mg/mL. The use of the enzymes from G. trabeum and T. emersonii extracts in mixtures with commercial Celluclast was based on the total loads of endoglucanases, since filter paper activities were not detectable in the extracts obtained from the studied fungi. Cellulose and xylan from the low recalcitrance substrate were converted at yields of 80% after 72 h of hydrolysis by Celluclast loaded at 120 UI of endoglucanases/g de substrate. Using enzyme mixtures with the same endoglucanase dosage but divided in 50% from Celluclast and 50% from G. trabeum extract yielded almost the same hydrolysis efficiency. In the case of the enzyme mixtures in which the extract from T. emersonii was used instead the extract from G. trabeum the hydrolysis efficiency was lower than that obtained by the treatment with Celluclast only. Cellulose and xylan from the low recalcitrance substrate were converted at yields of 50% after 72 h of hydrolysis by Celluclast. The replacement of this endoglucanase load with endoglucanases from G. trabeum or T. emersonii extracts resulted in lower hydrolysis efficiency.

Talaromyces versatilis est un champignon filamenteux d’intérêt industriel grâce à sa capacité deproduction d’enzymes hydrolytiques. La Société Adisseo commercialise un cocktail enzymatiqueproduit par fermentation à partir de T. versatilis, sous le nom de Rovabio™. Ce cocktail est utilisé entant qu'additif alimentaire en nutrition animale, car la grande variété d'enzymes hydrolytiques qu’ilcontient peut dégrader les polysaccharides présents dans l’enveloppe des céréales, améliorant ainsila digestibilité la valeur nutritionnelle des matières premières agricoles. Malgré les efforts consentispour mieux connaître la biologie de T. versatilis, très peu est connu sur ce champignon.L’étudeprésentée ici vise à décrire les réseaux de régulation qui contrôlent l’expression des gènes codantpour ces enzymes hydrolytiques, en utilisant des approches génomiques et transcriptomiques.Avoir accès à une annotation correcte de la séquence génomique et posséder les outilsnécessaires pour l'ingénierie génétique sont essentiels pour réaliser des études de génomiquefonctionnelle. Donc, le premier volet de cette thèse a été l’analyse de la séquence génomique et lacuration manuelle de l'annotation, ce qui nous a conduits à évaluer le vaste potentiel génétique de T.versatilis pour la production et la sécrétion d'enzymes hydrolytiques impliquées dans la dégradationde la lignocellulose. Deuxièmement, un système de délétion des gènes initialement conçu pourAspergillus niger a été adapté à T. versatilis. Cette méthode permet le recyclage du marqueur desélection et est efficace dans des souches dont le système NHEJ est actif (Delmas, et al., 2014, AEM).Au cours de ce travail, deux mutants de délétion de T. versatilis ont été obtenus: ΔxlnR et ΔclrA.La première approche mise en place pour avoir une meilleure compréhension des réseaux derégulation via une vue globale du transcriptome, fut l’utilisation de la technique de RNAseq sur troiséchantillons issus de la souche sauvage de T. versatilis exposée au glucose, à la paille de blé et auglucose et paille de blé simultanément comme sources de carbone, respectivement. Les données ontmontré une augmentation massive des niveaux d’expression de nombreux gènes, en particulier ceuxcodant pour des enzymes hydrolytiques, lorsque le mycélium est exposé à la lignocellulose. Enfin, la dernière partie du projet s’est appuyée sur la la RT-qPCR, technique appropriée pourétudier un nombre limité de gènes dans une grande variété de conditions. Toutefois la normalisationdes données est une étape essentielle du flux de travail qui peut conduire à une interprétationbiologique incorrecte de la régulation des gènes. Le travail effectué sur les données de RNAseq nousa amené à reconsidérer la nature des gènes de référence classiquement utilisés, puisque la plupartd'entre eux présentaient des changements d'expression considérables en présence de lignocellulose.En conséquence, un nouvel ensemble de gènes de référence putatifs a été identifié et la stabilité deleur expression validée par RT-qPCR chez T. versatilis cultivé dans plus de 30 conditions différentes.Des jeux de données de RNAseq de 18 champignons filamenteux phylogénétiquement éloignés ontpar ailleurs été collectés, afin de démontrer que la sélection des gènes candidats pour lanormalisation des données de RT-qPCR chez T. versatilis peut être étendue à d'autres champignons(Llanos et al., 2014, BMC Genomics). Ces aspects méthodologiques validés, nous avons enfin réaliséune étude plus détaillée de la transcription d'un groupe de gènes d'intérêt par RT-qPCR, dans unegrande variété de conditions et 2 souches différentes, la souche sauvage et la mutante ΔxlnR.L'analyse de ces données a permis d'identifier des gènes aux profils d'expression similaires, quirépondent de la même façon aux substrats inducteurs et qui partagent probablement les mêmesmécanismes de régulation. / Talaromyces versatilis is an industrially important enzymes producing filamentous fungus.Adisseo Company commercializes the enzymatic cocktail, produced from T. versatilis fermentation,with the name of Rovabio™. This cocktail is applied as an animal feed additive as it contains a widevariety of hydrolytic enzymes that can degrade the polysaccharides present in the seed-coat and thusimproves the digestibility and increases the nutritional value of the agricultural raw materials.Although efforts have been done to study different aspects of the biology of T. versatilis, very little isknown about this fungus. This study aimed to describe the regulatory networks of genes encodingplant cell wall-degrading enzymes from this biotechnologically important fungus using genomic andtranscriptomic approaches.Having a correct annotation of the genomic sequence together with efficient tools for genomeengineering are essential for downstream functional genomics works and characterization of theregulatory networks. Therefore, the first task carried out an analysis of the genomic sequence and amanual curation of the annotation, which led us to assess the vast genetic potential of T. versatilis forthe production and secretion of hydrolytic enzymes involved in the degradation of lignocellulosicmaterials. Secondly, I adapted a gene deletion system initially designed for Aspergillus niger. Thismethod allows recycling of the selection marker and is efficient in a non-homologous end-joining(NHEJ)-proficient strain (Delmas, Llanos et al., 2014, AEM). During this work, two deletion mutants ofT. versatilis were obtained: ΔxlnR and ΔclrA.Towards better understanding of the regulatory network, I first contributed to an RNAseq-basedtranscriptomic study that was performed on the wild type strain of T. versatilis exposed to glucoseand wheat straw as carbon sources. The data showed a massive increase in transcript levels ofnumerous genes, in particular those encoding hydrolytic enzymes, when the mycelium wasincubated with lignocellulose.If RT-qPCR is indeed a suitable technique to study a limited number of genes in a large variety ofconditions, data normalisation is a critical step of the workflow that can lead to incorrect biologicalinterpretation of gene regulation. The work done on the RNA-seq data led me to reconsider the useof the classical reference genes, since most of them exhibited expression changes in the presence oflignocellulosic substrate. I therefore identified a new set of putative reference genes and validatedtheir expression stability by RT-qPCR in T. versatilis cultivated under more than 30 differentconditions. Then, I collected about a hundred RNA-seq datasets from 18 phylogenetically distantfilamentous fungi, to demonstrate that the use of the suitable candidates for RT-qPCR datanormalisation in T. versatilis can be extended to other fungi (Llanos et al., 2014 BMC genomics (minorrevisions)). Thereafter, I performed a more detailed RT-qPCR based transcriptional study of a groupof genes of interest, in a wide variety of conditions and in 2 strains, the wild-type and the ΔxlnRmutant. The analysis of expression data of the genes of interest allowed to identify genes with similarexpression patterns, which probably share the same regulatory mechanisms and also the substratesthat act as inducers for their expression